<html><head><meta http-equiv="Content-Type" content="text/html charset=utf-8"></head><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;" class="">Should have said. The major trend in all Protostome evolution tends to be gene loss as opposed to gene gain. And the process of gene loss is more pronounced in the Ecdysozoa (insects and nematodes) than in the Lophotrochozoa (mollusks and flat worms).<div class=""><br class=""></div><div class="">—Carson</div><div class=""><br class=""><div><blockquote type="cite" class=""><div class="">On Aug 30, 2015, at 11:35 AM, Carson Holt <<a href="mailto:carsonhh@gmail.com" class="">carsonhh@gmail.com</a>> wrote:</div><br class="Apple-interchange-newline"><div class=""><meta http-equiv="Content-Type" content="text/html charset=utf-8" class=""><div style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;" class="">Sorry I meant to say Protostome not Deuterostomes (last sentence).<div class=""><br class=""></div><div class="">—Carson</div><div class=""><br class=""><div class=""><blockquote type="cite" class=""><div class="">On Aug 30, 2015, at 11:31 AM, Carson Holt <<a href="mailto:carsonhh@gmail.com" class="">carsonhh@gmail.com</a>> wrote:</div><br class="Apple-interchange-newline"><div class=""><meta http-equiv="Content-Type" content="text/html charset=utf-8" class=""><div style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;" class=""><div class="">Hi John,</div><div class=""><br class=""></div>MAKER requires that at least some form of evidence support each model. This is because ab initio predictors like Augustus and SNAP tend to over predict, in extreme cases by as much as 10 fold. So if you do not have any mRNA based evidence the chance that you would find something novel is extremely limited. This may be further complicated by the fact that nematodes tend to be highly divergent at the amino acid level, more than would be expected from their evolutionary relationship to other organisms. Their molecular clock is highly accelerated, and even making a phylogenic tree using nematodes is a nightmare because of the extremely long branch lengths they generate. So you may miss a lot of orthologs simply because proteins won’t align across such divergence. A classic example is smg-5 in C. elegans which will not align to its supposed orthologs in other eukaryotes at the amino acid level, but appears to have a conserved function and domain structure that is only detectable at a higher level.<div class=""><br class=""></div><div class="">You may be able to rescue a number of rejected gene models by using InterProScan to identify protein domains from the non-overlapping-ab-inits fasta files. In the absence of mRNA evidence, that really would be the best way of capturing as much as possible.</div><div class=""><br class=""></div><div class="">Also keep in mind that if the nematode you are working with is parasitic, then it’s evolution will be dominated by genome reduction as apposed to evolving new and novel genes. So having a smaller gene count with fewer novel genes will be expected. In fact the major trend in all Deterstome evolution tends to be gene loss as opposed to gene gain.</div><div class=""><br class=""></div><div class="">Thanks,</div><div class="">Carson<br class=""><div class=""><br class=""></div><div class=""><br class=""></div><div class=""><br class=""><div class=""><blockquote type="cite" class=""><div class="">On Aug 27, 2015, at 10:36 AM, John Bracht <<a href="mailto:johnbracht@gmail.com" class="">johnbracht@gmail.com</a>> wrote:</div><br class="Apple-interchange-newline"><div class=""><div dir="ltr" class=""><div style="font-size:12.8000001907349px" class="">Hi Carson, all,</div><div style="font-size:12.8000001907349px" class=""><br class=""></div><div style="font-size:12.8000001907349px" class="">I am a new user of Maker and have been overall quite pleased with it. Let me say also that the information I've gleaned on this forum has been extremely helpful and important in learning how to successfully use the software. I'm at a point where I'm planning next steps and hoping to solicit suggestions and ideas.</div><div style="font-size:12.8000001907349px" class=""><br class=""></div><div style="font-size:12.8000001907349px" class="">Briefly, I'm annotating a novel nematode genome that appears to have a reduced genome (~60 Mb, but about 24% repetitive, with a custom RepeatModeler library that we generated). For technical reasons I do not have access to RNA data so I'm running ab-initio predictors SNAP and Augustus supplemented with lots of protein hints: swiss-prot and a combined library of 28 nematode proteomes. </div><div style="font-size:12.8000001907349px" class=""><br class=""></div><div style="font-size:12.8000001907349px" class="">Maker Round 0: My student and I initially ran Maker without any active predictors, just to align the swiss-prot database to the genome (no nematode proteins used in this round), which output a .gff file with swiss-prot alignments, but no predicted proteins as SNAP and Augustus were turned off. We consider this 'round 0' of Maker.</div><div style="font-size:12.8000001907349px" class=""><br class=""></div><div style="font-size:12.8000001907349px" class="">Maker Round 1: We then trained SNAP with CEGMA output (which showed the assembly is 98% complete and identified 243 eukaryotic orthologs in our genome assembly). We proceeded with Maker round 1, run with CEGMA-trained SNAP, supplemented with a combined library of 28 nematode proteomes (protein predictions obtained from wormbase) and inputting the gff file containing the swiss-prot alignments from 'round 0' of Maker (protein_pass set to 1). Round 1 produced about 10,000 proteins.</div><div style="font-size:12.8000001907349px" class=""><br class=""></div><div style="font-size:12.8000001907349px" class="">Maker Round 2: The output of Round 1 was then filtered for protein hints giving a training set of about 3,000 proteins that we used in re-training SNAP, and in training Augustus. We then re-ran Maker without protein hints but with SNAP and Augustus re-trained; we did however feed in the .gff file produced in Round 1, containing all protein alignments (protein_pass set to 1). This gff file has all the swiss-prot alignments as well as the 28 nematode proteome alignments. Round 2 of Maker produced 9,374 proteins. At first I thought this was only half of what should be produced but now I'm re-evaluating that notion. I've been comparing known domains and protein families and the numbers are quite comparable across nematodes. There is a clear 'core' of conserved nematode proteins (about 5,000) and with C. elegans we identify 5,684 ortholog clusters by OrthoVenn (and OrthoDB). My student and I have manually inspected a number of predictions and they appear quite reasonable, with good numbers of introns etc (some introns are on the small side but that's also consistent with a reduced genome size). </div><div style="font-size:12.8000001907349px" class=""><br class=""></div><div style="font-size:12.8000001907349px" class="">My uncertainty arises from this: I took the 9,374 proteins and performed blastp against the 28 nematode proteome database from our training, and found that only 72 are 'novel', lacking a blast-match (e-value 1e-5). It is widely reported in the literature that new nematode projects often identify about 30% novel proteins with no blast-match to any other organism. I am concerned that we are missing these novel proteins, perhaps due to our heavy reliance on 'known' proteins as hints in Maker training. On the other hand, novel proteins could have been predicted as easily as other proteins by Maker, as to my knowledge there is no requirement that protein hints support a given prediction. (About 18% of our proteins have no Pfam domain predictions, so in some sense they are novel, but they're matching other 'unknown' proteins in other nematodes by blast). </div><div style="font-size:12.8000001907349px" class=""><br class=""></div><div style="font-size:12.8000001907349px" class="">In sum, I'm wondering if we should re-evaluate some step of our Maker pipeline or whether we are likely to be on safe ground concluding that the 9,374 is relatively representative of the full proteome of this organism. Interestingly, given the small genome size, there simply isn't much room for more proteins to be predicted--once you account for the high repetitive nature of this genome the gene density is slightly higher than that of C. elegans, C. briggsae, and P. pacificus. Looking at the scaffolds, they are quite densely populated with predictions and there are not obvious 'gaps' where new predictions might be derived. From these observations I am tending toward the idea that the 9,374 proteins is relatively complete and that a lack of novel proteins is actually a scientific finding in this organism (it lives in an unusual environment where this might make sense) but I'd like to be sure we are seeing a real phenomenon, and not some weird artifact of the way we predicted the proteins.</div><div style="font-size:12.8000001907349px" class=""><br class=""></div><div style="font-size:12.8000001907349px" class="">For the record, the N50 of our assembly is about 50kb, so I don't think we're missing a lot of genes due to fragmentation, and at any rate that shouldn't preferentially affect 'novel' genes more than 'known' genes.</div><div style="font-size:12.8000001907349px" class=""><br class=""></div><div style="font-size:12.8000001907349px" class="">So: where are the novel proteins? Should we amend our Maker pipeline? Thanks for any an all ideas, questions, or comments! I'm attaching our maker_opts.ctl file from the last run (Round 2) in case it is helpful.</div><div style="font-size:12.8000001907349px" class=""><br class=""></div><div style="font-size:12.8000001907349px" class="">Thanks,</div><div style="font-size:12.8000001907349px" class="">John</div></div>
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